This invention relates to a novel human neurotensin receptor, designated HNT2R, present in two isoforms, a long isoform designated HNT2RL, and a deletion variant (short) form of the long isoform designated HNT2RS, and to nucleic acids encoding the human neurotensin receptor proteins. The invention further relates to high throughput screening assays with these receptors.
Neurotensin is a neuropeptide that is found predominantly in the hypothalamus and gut. Neurotensin acts as a neurotransmitter and neuromodulator in the brain and as a hormone in the periphery. Native neurotensin [NT (1-13)] is a 13 amino acid peptide. However, the carboxyl terminal 6 amino acids of neurotensin (8-13) [NT (8-13)] and the more stable N-acetylated form [AcNT (8-13)] are sufficient to elicit fill biological activity mediated by neurotensin in most, but not all, tissues. A closely related peptide, neuromedin N, also binds to and activates neurotensin receptors, but with lower affinity than neurotensin. Neurotensin and neuromedin N are processed from the same precursor protein.
An important response mediated by neurotensin is suppression of appetite, making this hormone a potential anti-obesity target. Both central and peripheral administration of neurotensin suppresses appetite in fed and fasted animals without altering water consumption or initiating a conditioned taste aversion (Stanley et al., Peptides 4:493-500, 1983; Hawkins, Physiol. and Behavior 36:1-8, 1986; Luttinger et al., Eur. J. Pharmacol. 81:499-503, 1982; Bailey and Flatt, Comp. Biochem. and Physiology, 84:451-4,1986). The active moiety of neurotensin is only 6 amino acids, making this potential target amenable to high throughput screens for the discovery of non-peptide agonists. Very little has been reported, however, on the chronic effects of neurotensin on metabolic responses in vivo.
Obesity is a complex, multifactorial disease defined as a body mass index (BMI) of greater than 27 kg/m2 (relative weight xe2x89xa7120% of normal). Morbid obesity is defined as a BMI of 40 kg/m2 (relative weightxe2x89xa7200% of normal) or greater. Many genetic and environmental factors can lead to the development of obesity, including decreased resting metabolic expenditure and hyperphagia. A significant number of obese subjects have characteristic metabolic changes associated with this disease. Many are hyperinsulinemic as well as hyperglycemic indicating an insulin-resistant state, a common characteristic of non-insulin dependent diabetes mellitus (NIDDM).
The ideal anti-obesity agent would have to accomplish several tasks for it to be successful therapeutically. Three of the most important effects mediated by effective anti-obesity agents would be to increase satiety, increase metabolic energy expenditure, and increase utilization of existing fat depots (lipolytic). Most individual medical treatments for obesity either increase metabolic energy expenditure (xcex23-agonists) or decrease appetite (dexfenfluramine). Very few drugs actually do both. In fact, many appetite suppressants cause a decrease in metabolic energy expenditure to compensate for the decreased energy intake. Thus, there is a need for a novel anti-obesity target that increases energy expenditure as well as decreases appetite.
In animal models of obesity and in human obese patients, neurotensin levels are decreased when compared to lean animals and humans. Decreased hypothalamic neurotensin concentrations (up to 50%) have been observed in five different animal models of obesity: ob/ob, db/db, and CPE mice, and fa/faZucker and Corpulent rat models (Beck et al., Neuropeptides, 13:1-7, 1989; Williams et al., Metabolism 40:1112-16. 1991, Beck et al., J. Nutrition, 120:806-811, 1990). For ob/ob mice, the decreased hypothalamic neurotensin concentration precedes the onset of non-insulin-dependent diabetes. Decreased plasma neurotensin levels have also been observed in diabetic obese, but not diabetic lean, humans suggesting that insulin does not play a direct role in the regulation of neurotensin levels or signaling (Service et al., Regulatory Peptides 14:85-92, 1986). However, neurotensin may play a role in the regulation of insulin secretion, as high-affinity neurotensin binding sites are found in the pancreas. More importantly, the increase in plasma neurotensin levels observed in response to a meal is not observed in obese humans (Bloom et al., Anal. NY Acad. Sci. 400:105-114, 1982). Exogenously administered neurotensin suppresses appetite in lean, ob/ob, and db/db mice, suggesting that even though this important satiety signal is decreased in obesity, the signal transduction pathway is still fully functional in obese animal models.
In vivo studies measuring the effects of AcNT(8-13) on modulation of appetite were conducted with several different mouse models of obesity. AcNT(8-13) i.p. was shown to promote biphasic suppression of food consumption in fasted ob/ob males. It was demonstrated that in animals pretreated with the NT2R-selective antagonist levocabastine, the levocabastine completely reversed the suppression of food intake mediated by 50 xcexcg/kg AcNT(8-13), suggesting that an NT2R-like subtype mediates the NT response of feeding.
To determine whether blockade of the neurotensin response is a general phenomenon among antihistamines or specific to levocabastine, a second antihistamine, pyridine, was also tested. Pyridine exhibits high affinity binding to H1-histamine receptors but not to any NTR subtypes. Pyridine (1 mg/kg) did not block the AcNT(8-13)-mediated appetite suppression. These data indicate that the AcNT(8-13)-mediated blockade of appetite suppression by levocabastine is NTR-selective and not a general response to antihistamines.
Obesity is associated with decreased metabolic energy expenditure and increased metabolic efficiency. Increasing metabolic energy expenditure (thermogenesis) is a desirable characteristic of anti-obesity targets. AcNT(8-13), when administered acutely, displays many of the characteristics required for a good anti-obesity target, such as: appetite suppressionxe2x80x94food consumption is suppressed 40-60%; lipolytic effectsxe2x80x94elevated NEFAs and glycerol are observed in response to AcNT(8-13); and oxygen consumption is increasedxe2x80x94thermogenesis is controlled by the activity of a family of proteins termed uncoupling protein (UCP); UCP uncouples respiration leading to generation of heat. Thus, there is a need in the art to identify neurotensin agonists, particularly agonists that are selective for the subtype 2 neurotensin receptor.
Based on the use of selective antagonists, three neurotensin receptor subtypes have been identified. These are designated NT1R, NT2R, and NT3R. Pharmacological data support the existence of a separate neuromedin N receptor. Thus, a fourth subtype with high affinity for the closely related peptide neuromedin N has also been proposed. Two of these receptor subtypes, NT1R and NT2R (termed high and low affinity NT binding sites), have been identified in rat by molecular cloning. All four subtypes are members of the G protein-coupled receptor superfamily and display many of the structural characteristics of this receptor family. G protein-coupled receptors, characterized by seven transmembrane domains, mediate many extracellular signals and are present in organisms as divergent as yeast and man.
Rat NT1R is a 424 amino acid polypeptide (Tanaka et al., Neuron 4:847-854, 1990; Vita et al., FEBS Letters 317:139-142, 1993). The quinoline pyrazole antagonist, SR48692 ({2-[1-(7-chloro-4-quinolinyl)-5-(2,6-dimethoxyphenyl)pyrazol-3-yl)carbonylamino]tricyclo (3,3,1,1,3,7)-decan-2-carboxyic acid}), displays a 20 to 100-fold selectivity for the NT1R over the NT2R subtype. The NT1R subtype appears to couple to at least three G protein-mediated pathways, namely, release of intracellular calcium, release of cGMP-dependent protein kinase, and modulation of adenyl cyclase activity.
Rat NT2R is a 416 amino acid polypeptide (Chalon et al., FEBS Letters 386:91-94, 1996; Marzella et al., J. Neuroscience 16:5613-20, 1996), which is 43% identical and 64% homologous to the NT1R subtype. The phenyl piperidine antagonist levocabastine (Jansenn, (xe2x88x92)-{3S-[1(cis)-3xcex14xcex2]}-1-{4-cyano-4-(4-fluoro-phenyl)-cyclohexyl)}-3-methyl-4-phenyl-4-piperidene-carboxylic acid monohydrochloride) binds with high affinity to the NT2R subtype, and with high selectivity versus the NT1R subtype. Levocabastine exhibits a 1000-fold greater selectivity for the NT2R over the NT1R and NT3R subtypes. Levocabastine is also an antihistamine.
European Patent Publication 875 568 A1(published application) reports isolation of a putative human neurotensin receptor type 2 having 319 amino acids. Alternate splice forms yielding shorter polypeptides were also reported. The amino acid sequence reportedly was 86.86% identical (Using Bestfit) with rat NT2R (citing Chalon et al., supra for the rat NT2R). The nucelotide sequence was reported to be 79.0% identical (using Bestfit) to the rat sequence.
Little is known about the signal transduction pathways mediated by the NT2R subtype. Some neurotensin analogs elicit biological responses that cannot be ascribed to either the NT1R or NT2R subtypes. By process of elimination (responses that are not blocked by levocabastine or by SR48692), a third NT subtype has been proposed, NT3R. A third antagonist, SR142948 (2-{[5-(2,6-dimethoxyphenyl)- 1-(4-(N-(3-dimethylaminopropyl)-N-methyl-carbamoyl)-2-isopropylphenyl-1H-pyrazole-3-carbonyl]-amino}adamantane-2-carboxylic acid, hydrochloride) is an antagonist that shows comparable affinity for the NT1R and NT2R subtypes but may also bind to the neuromedin N receptor (Gully et al., JPET, 280:802-812, 1997). All three antagonists display good in vivo efficacy and can be used to define the neurotensin receptor subtypes mediating physiological responses ascribed to neurotensin.
Both NT1R and NT2R are found in the primary feeding control center, the hypothalamus. NT2R messenger RNA is approximately 100-fold more abundant than NT1R mRNA in the hypothalamus. Use of SR48692 and levocabastine in vivo and in vitro can discriminate between NT1R and NT2R-mediated events.
In U.S. Pat. No. 5,668,006, G-protein linked receptors are reported to control many physiological functions, such as mediating transmembrane signaling from external stimuli (vision, taste and smell), endocrine function (pituitary and adrenal), exocrine function (pancreas), heart rate, lipolysis, and carbohydrate metabolism. The molecular cloning of a number of such receptors have revealed many structural and genetic similarities, permitting classification of the G protein-linked receptor superfamily into five distinct groups.
U.S. Pat. No. 5,691,188, describes how upon binding to the receptor, the receptor presumably undergoes a conformation change leading to activation of the G protein. G proteins are described as being comprised of three subunits: a guanyl-nucleotide binding a subunit; a xcex2 subunit; and a xcex3 subunit. G proteins cycle between two forms, depending on whether GDP or GTP is bound to the xcex1 subunit. When GDP is bound, the G protein exists as a heterotrimer, the Gxcex1xcex2xcex3 complex. When GTP is bound, the xcex1 subunit dissociates, leaving a Gxcex2xcex3 complex. When a Gxcex1xcex2xcex3 complex operatively associates with an activated G protein coupled receptor in a cell membrane, the rate of exchange of GTP for bound GDP is increased and the rate of dissociation of the bound Gxcex1 subunit from the Gxcex1xcex2xcex3 complex increases. The free Gxcex1 subunit and Gxcex2xcex3 complex are capable of transmitting a signal to downstream elements of a variety of signal transduction pathways. This fundamental scheme of events forms the basis for a multiplicity of different cell signaling phenomena.
In response to binding of neurotensin, the neurotensin receptor activates a G protein, which in turn modifies the activity of a variety of effector proteins. As with previously cloned receptors, hydropathy analyses of human and rat NT1R and rat NT2R sequences show seven distinct hydrophobic domains, each of which are thought to traverse the cell membrane. Both subtypes are similar in overall size, but their sequences display several differences. The amino terminus of NT1R is 33 residues longer than that of NT2R, and the third intracellular loop of NT2R is 20 residues longer than that of NT1R. The third intracellular loop of G protein coupled receptors is thought to contain sites for interaction with G proteins, thereby providing a mechanism by which extracellular signals are transduced into the cell. The differences in size as well as sequence within this loop suggest that NT1R and NT2R may couple to different G proteins, may interact differently with the same G protein, or both. The transmembrane domain 2aspartate found in many G protein-coupled receptors confers sodium sensitivity as well as coupling to certain effectors. Each of the extracellular loops of both receptor subtypes contain a cysteine residue. The DRY sequence common to many receptors, however, is an ERY in NT1R and an ERC in NT2R. The intracellular carboxyl terminus of each subtype contains a palmitoylation site as well as several serine and threonine residues, which may serve as potential phosphorylation sites.
In one embodiment, the invention provides an isolated nucleic acid comprising a sequence that encodes a functional human neurotensin subtype 2 receptor (HNT2R). In specific embodiments, the invention provides a long isoform and a short isoform of the receptor. The invention further provides vectors and host cells containing such vectors, as well as methods for expressing the HNT2R.
In a further embodiment, the invention provides an isolated nucleic acid of at least about 15 nucleotide bases that hybridizes under highly stringent conditions with a nucleic acid encoding the NTR2R.
In yet a further embodiment, the invention provides an isolated human neurotensin subtype 2 receptor (HNT2R), including both the long and short isoforms thereof. Also provided is an antibody that specifically binds to the HNT2R.
In yet another embodiment, the invention provides a method for identifying a compound that binds to HNT2R comprising detecting binding of a test compound to HNT2R.
For the first time, functional cDNA clones encoding the entire HNT2R receptor have been isolated and their expression characterized. Surprisingly, two functional isoforms have been found: HNT2RL (long), which appears to be the full length receptor polypeptide, and HNT2RS (short), which is a deletion variant (splicing variant) form of HNT2RL. Relative to HNT2RL (long), HNT2RS (short) lacks 58 nucleotides in the region encoding IC3 and TM6. The existence of HNT2RS was completely unexpected; no rat or other homolog of this isoform has been reported to date.